This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Centrioles are organelles that play essential roles in the formation of cilia and flagella, and they participate in cytokinesis, cell cycle control and centrosome organization. The cell's single centrosome, which is inherited as a spindle pole, duplicates only once per cell cycle, a process that is regulated through the control of centriole duplication. Centriole duplication is strictly controlled to ensure the formation of a bipolar spindle and the maintenance of ploidy. It has been known for many years that daughter centrioles assemble at right angles to the older, or "mother" centriole. The significance of this and the pathway for daughter centriole assemble remain mysterious. We are using the correlative light and EM tomography approach to study centrosome assembly in staged C. elegans embryos. Our initial results showed that daughter centriole assembly in this organism begins with the formation of a central tube, just as the pronuclei appear (Pelletier, L., O'Toole, E.T., Schwager, A., Hyman, A.A., and M[unreadable]ller-Reichert, T. (2006) Centriole assembly in Caenorhabditis elegans. Nature 444, 619-23). The central tube elongates during pronuclear migration, and the assembly of singlet microtubules on the tube occurs during pronuclear rotation. Using EM-tomography in combination with RNA interference and restoration microscopy we have found evidence suggesting that tube formation and elongation requires the SAS-5/SAS-6 proteins and an upstream signal mediated by SPD-2 and ZYG-1. We further show that although the assembly of the central tube still occurs, singlet microtubules fail to assemble in the absence of SAS-4, suggesting that it is required specifically at that step of assembly. The centriole assembly pathway we describe here in C. elegans may provide a framework for further studies on centriole assembly in other centriole bearing organisms.